In article <199404141123.UAA14023 at inetnif.niftyserve.or.jp>,
HGF02633 at niftyserve.or.jp (=?ISO-2022-JP?B?GyRAQFBETT1TPCMbKEo=?=) wrote:
>> Does anybody know sesitive assay system to detect beta-galactosidase
> activity in cell extracts after DNA transfection (lipofectin method).
> We transfected pCH110 (beta-galactosidase expression vector) into PC12
> cells. However, the transfection efficiency was very low. We could not
> detect the enzyme activity with o-nitrophenol-b-D-galactopyranoside
> (ONPG) as a substrate, though the CAT activity was detected in the same
> experiment. How can we overcome this problem. please teach me.
>The Galacto-Light kit from Tropix (FAX: (617)275-8581 US) is exquisitely
sensitive (cf Jain and Magrath Anal. Biochem 199:119-124) to beta-Gal. It
is much cheaper to make the solutions yourself but as a tryout the kit is
reasonably priced. You will need a luminometer or a scintillation counter
in out-of coincidence mode.
The main problem with beta-gal is from endogenous lysosomal beta-Gal
activity and the solution used by Jain and Magrath doesn't solve it
completely in cells with high endogenous background. I use a modification
incorporating a heat inactivation step (Young et al Anal Biochem
215:24-30). So harvesting the cells in the buffer and heat-inactivating as
in the Young paper and doing the beta-Gal assay as in the Jain paper works
a treat. I also use some of the uninactivated extract for luciferase
assays.
In fact, if you combine the secreted alk phos, human growth hormone,
luciferase and beta-Gal assays, you can theoretically use 3 reporters and
still have one transfection control!!!!!!
David Huen, Institute of Cancer Studies, Univ. of Birmingham, U.K.